Use of an in vitro hemodynamic endothelial/smooth muscle cell co-culture model to identify new therapeutic targets for vascular disease

ABSTRACT

Methods and devices for applying hemodynamic patterns to human/animal cells in culture are described. Hemodynamic flow patterns are measured directly from the human circulation and translated to a motor that controls the rotation of a cone. The cone is submerged in fluid (i.e., cell culture media) and brought into close proximity to the cells. Rotation of the cone creates time-varying shear stresses. This model closely mimics the physiological hemodynamic forces imparted on endothelial cells in vivo. A TRANSWELL coculture dish (i.e., a coculture dish comprising an artificial porous membrane) may be incorporated, permitting two, three, or more different cell types to be physically separated within the culture dish environment. In-flow and out-flow tubing may be used to supply media, drugs, etc. separately and independently to both the inner and outer chambers. The physical separation of the cell types permits each cell type to be separately isolated for analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/007,483, now U.S. Pat. No. 7,811,782, filed on Jan. 10, 2008, whichclaims priority under 35 U.S.C. §119(e) from U.S. Provisional PatentApplication No. 60/879,710, filed on Jan. 10, 2007, which isincorporated herein by reference.

STATEMENT REGARDING SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO SEQUENCE LISTING

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to devices and methods for in vitroanalysis of fluid flow (e.g., hemodynamics) on cells (e.g., endothelialcells). More specifically, this invention relates to a method of using adevice that permits more than one different cell types to be physicallyseparated within the culture dish environment, while the inner cellularsurface is exposed to the simulated hemodynamic flow patterns.

2. Description of Related Art

Atherosclerosis is a vascular inflammatory disease characterized bylesion formation and luminal narrowing of the arteries. Endothelial cell(EC) and smooth muscle cell (SMC) regional phenotypes have significantimplications in the progression of vascular disease. During earlyatherogenesis, the endothelium becomes activated, leading to increasedadhesion molecule expression, permeability to lipoproteins and cytokinegeneration. Such environmental changes can influence SMCs to undergo“phenotypic switching” characterized by morphological changes, increasedproliferation and migration, and decreased expression of definingquiescent SMC markers.

Atherosclerosis is further characterized by its focal development inlarge arteries at hemodynamically defined regions, such as atbifurcations that produce complex flow patterns. Atheroprone regions,susceptible to plaque formation, are subjected to low time-averagedshear stress and “disturbed” oscillatory flow patterns. In contrast,atheroprotective regions, which are less susceptible to plaqueformation, are exposed to relatively higher time averaged shear stressand pulsatile laminar flow (13, 39). In regions of chronic disturbedflow, changes in EC phenotype, such as increased adhesion moleculeexpression, (i.e., vascular cell adhesion molecule 1 (VCAM-1),intercellular adhesion module 1 (ICAM-1), e-Selectin), andtransendothelial permeability to low density lipoproteins (LDL), willeffect the local signaling environment and can alter SMC phenotype,leading to proliferation, migration and the pathogenesis ofatherosclerosis.

The factors controlling changes in SMC phenotype involving EC's andhemodynamic flow patterns are not fully understood. However, a hallmarkof SMC phenotype switching in atherosclerosis is the suppression ofcontractile proteins that define the differentiated SMC, includingSMMHC, SMαA, and myocardin.

To understand the role of shear stress on the endothelium inatherogenesis, in vitro models that expose ECs to a variety of shearstress conditions have been extensively studied. Since ECs candiscriminate variations in flow patterns and are sensitive to both shearstress magnitude and time-varying features of hemodynamics, emulating invivo flow environments appears to have a greater impression onrecapitulating the in vivo phenotype of the endothelium. Additionally,few studies have shown the intricate interactions andcross-communications of ECs and SMCs in the presence of any type offlow, and no known studies to date have examined how in vivo-derivedhuman hemodynamic forces on the endothelium regulate SMC phenotypicswitching, as it is classically defined by the literature.

SUMMARY OF THE INVENTION

An aspect of the invention is, but not limited thereto, an in vitrobiomechanical model used to apply hemodynamic (i.e., blood flow)patterns modeled after the human circulation to human/animal cells inculture. This model replicates hemodynamic flow patterns that aremeasured directly from the human circulation using non-invasive magneticresonance imaging and translated to the motor that controls the rotationof the cone. The cone is submerged in fluid (i.e., cell culture media)and brought into close proximity to the surface of the cells that aregrown on the plate surface. The rotation of the cone transduces momentumon the fluid and creates time-varying shear stresses on the plate orcellular surface. This model most closely mimics the physiologicalhemodynamic forces imparted on endothelial cells (cell lining bloodvessels) in vivo and overcomes previous flow devices limited in applyingmore simplified nonphysiological flow patterns.

Another aspect of this invention is directed to incorporate acommercially available TRANSWELL (a coculture dish comprising anartificial porous membrane), for example a 75 mm-diameter TRANSWELL.This permits two, three, or more different cell types to be physicallyseparated within the culture dish environment, while the inner cellularsurface is exposed to the simulated hemodynamic flow patterns. Othersignificant modifications include in-flow and out-flow tubing to supplymedia, drugs, etc. separately and independently to both the inner andouter chambers of the coculture model. External components are used tocontrol for physiological temperature and gas concentration. Thephysical separation of adherent cells by the artificial TRANSWELLmembrane and the bottom of the Petri dish permits each cell layer, orsurface, to be separately isolated for an array of biological analyses(i.e., protein, gene, etc.).

The directed use of this invention includes 1) to study the cross-talkbetween human/animal endothelial and smooth muscle cells—two criticalcell types that comprise the blood vessel wall and involved in thepathological development of atherosclerosis (heart disease, stroke,peripheral vascular disease) and other vascular diseases. 2) This modelmay also be used as a diagnostic model in testing novel drug-basedtherapies for toxicity, inflammation (e.g. monocyte adhesion,inflammatory cytokine release, inflammatory gene induction) andpermeability.

Some exemplary novel aspects of various embodiments related to thisinvention include, but not limited thereto, the following, in nospecific order:

The device can replicate with the highest level of fidelity thehemodynamic shear stress profiles in the arterial circulationsusceptible to and protective of atherosclerosis and from patientssusceptible to other physiological (e.g., exercise) or pathologicalconditions (e.g., hypertension, diabetes, dyslipidemia).

The device can replicate with the highest level of fidelity any type ofmeasurable or idealistic shear stress profiles from the arterial,venous, or any organ circulation.

Exposure of the hemodynamic flow patterns on the inner surface of aTRANSWELL membrane, with or without another cell type cultured on theopposing side of the membrane.

Exposure of the hemodynamic flow patterns on the inner surface of aTRANSWELL membrane, with or without another cell type cultured on thebottom surface of the TRANSWELL dish.

Exposure of the hemodynamic flow patterns on the inner surface of aTRANSWELL membrane, with or without another cell type cultured on theopposing side of the membrane and with or without a third cell typecultured on the bottom surface of the TRANSWELL dish. The third cell mayinclude monocytes or macrophages for inflammatory cell adhesion assays.

Exposure of the hemodynamic flow patterns on the inner surface of aTRANSWELL membrane, with or without another cell type cultured on theopposing side of the membrane and with or without a third cell type insuspension in the media of the inner surface of the TRANSWELL membrane.

Clamps mount on the sides of the TRANSWELL used to hold in place theinflow and out-flow tubing for both the inner (upper) chamber and outer(lower) chamber. This is used to perfuse in and out media, biochemicalcompounds agonist, antagonists, etc of the upper and/or lower chamber ofthe TRANSWELL separately without disturbing the flow environment. Mediaextracted from the experiment can be used to further test cytokine orhumoral factor secretion from either layer.

The ability to isolate each cell type independently (one, two, or threedifferent cell types used) from a single experiment for post-processingbiological (proteomic/genomic) analyses, including gene arrays,proteomics.

The device can accept and test any cell type from any species that isadherent or nonadherent.

The device can be used as a vascular biomimetic cell culture model forinvestigating all phases from embryonic vascular development to thesevere cases of atherosclerosis in adults. For example, endothelialcells may be plated in the inner surface and/or smooth muscle cellsplated on the opposing side of the TRANSWELL membrane and/or macrophages(or leukocytes) in the upper or lower chamber.

The device can be used to test the compatibility, cellular adhesion, andphenotypic modulation of cells from vascular stent material underhemodynamic conditions. For example, endothelial and/or smooth musclecells may be seeded next to, on top of, or underneath the material,mounted on the stationary surface of the device. Materials include butare not limited to metallic nanoporous metals, polymers, biodegradablepolymers, carbon surfaces, scratched or etched surfaces.

The device can be used to test drug (i.e., compound) elution fromvascular stent material under hemodynamic conditions in the presence orabsence of cells.

The device can be used to test the compatibility, cellular adhesion, andphenotypic modulation of cells seeded on or adjacent to surfaces coatedwith polymeric material under hemodynamic conditions.

The device can be used as a vascular biomimetic cell culture model forthe investigation of the blood-brain barrier. For example, endothelialcells may be plated in the inner surface and/or glial cells and/orastrocytes and/or neurons plated on the opposing side of the TRANSWELLmembrane and/or the bottom Petri dish surface.

The device can be used as an airway biomimetic cell culture model forthe investigation of the development and progression of asthma. Forexample, epithelial cells may be plated in the inner surface and/orsmooth muscle cells plated on the opposing side of the TRANSWELLmembrane and/or macrophages (or leukocytes) in the lower chamber.Rhythmic breathing patterns are emulated by the movements of the cone inclose approximation to secrete and/or artificial mucosal layer betweenthe cone and epithelial surface.

The device can be used as renal biomimetic model for the investigationendothelial cell and epithelial podocyte interaction.

The device can be used to create a specific humoral environment thatmimics patient drug therapy and then determine compatibility of a knownor unknown drug compound in conjunction with the patient drug therapy.For example, the device can be run for a specific time with the drugLIPITOR (atorvastatin) in the media and then an unknown drug can beadded to determine changes in toxicity, inflammation (e.g. monocyteadhesion, inflammatory cytokine release, inflammatory gene induction)and permeability.

The device can be used to determine functional changes in vascular cellsor other organ cells types taken from patients with an identifiedgenotype linked to drug toxicity or some pathophysiological endpoint.For example, endothelial cells from a patient with a single nucleotidepolymorphism (SNP) identified to be associated with drug toxicity can beused to test novel or known compounds for changes in toxicity,inflammation (e.g. monocyte adhesion, inflammatory cytokine release,inflammatory gene induction) and permeability. This is commonly referredto as pharmacogenomics.

An embodiment of this invention is a method of applying hemodynamicpatterns to cells in culture, said method comprising the steps ofplating a first set of cells on a TRANSWELL, plating a second set ofcells on said TRANSWELL, wherein said first set of cells are separatedfrom said second set of cells, adding a fluid to said TRANSWELL; andcausing rotation of said fluid for a period of time, wherein said mediumthus exerts a shear force upon said second set of cells.

Another embodiment of this invention is a method of applying hemodynamicpatterns to cells in culture, said method comprising the steps ofmonitoring the hemodynamic pattern of a subject; modeling saidhemodynamic pattern into a set of electronic instructions; and using adevice to cause a shear stress on a plurality of sets of cells on aTRANSWELL based upon said electronic instructions.

Another embodiment of this invention is a hemodynamic flow device,comprising an electronic controller; a motor, wherein said motor isoperated via said electronic controller; a cone connected to said motor,whereby said cone is rotated by said motor; a TRANSWELL with a membrane,wherein said cone is at least partially submerged in a medium in saidTRANSWELL and wherein said cone exerts a rotational force upon saidmedium; an inlet flow tube to add media to said TRANSWELL; and an outletflow tube to withdraw media from said TRANSWELL.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides an exemplary view of EC/SMC plating on a TRANSWELL;

FIG. 2 provides a view of the cone and plate flow device, modified toaccommodate a TRANSWELL culture dish;

FIG. 3 provides a graph displaying an exemplary hemodynamic flow patternderived from an MRI of a the human common carotid artery (CCA) andinternal carotid sinus (ICS). Also shown is such an exemplary MRI;

FIG. 4 shows exemplary confluent layers of ECs and SMCs twenty-fourhours following EC seeding;

FIG. 5 shows exemplary transverse sections stained for F-actin and FM4-64 or visualized by differential interference contrast showingcellular processes within membrane pores;

FIG. 6 shows exemplary immunofluorescence images of EC/SMC morphologyand orientation;

FIG. 7 shows exemplary normalized histogram plots of shape factors (SF)for ECs and SMCs;

FIG. 8 shows average angles of direction for SMCs and ECs relative tothe direction of atheroprotective flow (0°);

FIG. 9 shows orientation histograms of SMC direction (or angle) relativeto the direction of flow;

FIG. 10 shows an exemplary graph demonstrating normalized geneexpression;

FIG. 11 shows an exemplary graph of normalized mRNA expression;

FIG. 12 shows the results of an exemplary protein analysis;

FIG. 13 shows an exemplary graph of normalized mRNA expression;

FIG. 14 shows an exemplary graph of normalized mRNA expression;

FIG. 15 shows the results of an exemplary blot analysis;

FIG. 16 shows the results of an exemplary ELISA analysis for IL-8performed on atheroprone and atheroprotective flow-conditioned media;

FIG. 17 shows an exemplary graph of normalized mRNA expression;

FIG. 18 shows an exemplary scanning electron micrograph of the surfacesof the membrane; and

FIG. 19 shows a graph of exemplary fold enrichment.

DETAILED DESCRIPTION OF THE INVENTION Atherosclerosis

Atherosclerosis preferentially develops at arterial regions, such asbifurcations and regions of high curvature, characterized by disturbed,low time averaged and oscillatory wall shear stress. Atheroprone regionsin vivo and atheroprone shear stress on the endothelium in vitro caninduce proinflammatory priming indicated by the activation andregulation of downstream inflammatory targets. Although ECs and SMCs aretwo major cell types known to undergo phenotypic modulation, or“switching,” during initiating atherosclerotic events, until thisinvention it was unknown whether hemodynamic forces on ECs regulated orcontributed to this process in SMCs. Human-derived atheroprone shearstresses applied to ECs modulate a proinflammatory phenotype in ECs andSMCs and proatherogenic phenotypic switching in SMCs via epigeneticmodifications at the chromatin level. This is a process referred to asmechanotranscriptional coupling.

Results from the present coculture process support the hypothesis thathemodynamics induce vascular EC and SMC priming toward a proatherogenicresponse, thus validating the use of the coculture system as a newphysiologically relevant biomimetic vascular model for the study ofearly atherosclerotic events. These results are consistent withpreviously published atherosclerosis-related in vivo and in vitro flowstudies (see FIG. 10). Moreover, previous TRANSWELL coculture models ofECs and SMCs have been restricted to static-type experiments, with theexception of a few flow studies, and no known studies have employedphysiologically relevant, human-derived hemodynamic flow patterns. Thepresent process overcomes these limitations by directly comparing twohemodynamic flow patterns, yielding a more physiologically relevantmodel for accurately comparing in vivo regions in the vasculature, andfocused on classic SMC differentiation markers.

A hallmark of SMC phenotypic modulation in vascular disease is alteredexpression of genes that define the contractile phenotype. SMCdifferentiation markers and transcription factors that are delineatorsof a differentiated SMC are affected by atheroprone flow. The loss ofexpression of differentiation markers (SMαA and myocardin) and inductionof the inflammatory marker VCAM-1 at both mRNA and protein levelsconfirmed that ECs exposed to atheroprone flow differentially regulatethe SMC phenotype compared with atheroprotective flow. ChIP analysisrevealed that the mechanism initiating atheroprone-induced loss ofCArG-dependent SMC gene expression involved reduction of SRF binding toCArG box regions of SMαA and SMMHC and deacetylation of histone H4compared with atheroprotective flow. This was not the case for the earlygrowth response gene c-fos. These results are consistent with amonoculture SMC study in response to PDGF-BB treatment and, mostimportantly, the epigenetic fingerprint for SMαA, SMMHC, and c-fos inintact blood vessels in response to acute vascular injury. Thus, thegeneral paradigm that histone H4 acetylation is critical for maintainingCArG chromatin promoter regions in a SRF-accessible state isdifferentially regulated by two distinct hemodynamic flow patternsexposed to ECs. The SRF coactivator myocardin plays a critical role informing a higher-order complex with SRF for the positive regulationSMCselective CArG-dependent genes. In contrast, KLF4 can abrogatemyocardin-dependent regulation of CArG-dependent SMC differentiationgenes. Myocardin expression was significantly reduced in response toatheroprone flow, whereas KLF4 tended to have increased expression.Since KLF4 gene expression can be rapidly and transiently induced inresponse to PDGF-BB in cultured SMCs and transiently induced in intactvessels following acute vascular injury up to six hours and returning tobaseline by twenty-four hours, it is possible that the maximal and mostsignificant changes in KLF4 expression were not captured at this timepoint. Nevertheless, gene profiles generated in this study correlatewith existing data from the literature, and, taken together, the resultssuggest that phenotypic modulation of SMCs exposed to atheroprone flowoccurs at the transcriptional level and involves the well-characterizedSRF/myocardin and KLF4 signaling axis.

Of interest, ECs exhibited reduced KLF4 expression in atheroprone flow.KLF4 has been shown to be regulated by flow in ECs in monoculture;however, it was previously not known that KLF4 is differentiallyexpressed by atheroprone flow compared with atheroprotective flow. Thefunctional significance of KLF4 in ECs has recently been shown to besimilar to that of KLF2 (i.e., anti-inflammatory, atheroprotective, andhemostasis control). Moreover, KLF4 has been implicated in cell cycleregulation, and greater cell cycle activity has been reported foratheroprone relevant flow in vitro and regions in vivo. Thus, theregulation of KLF4 transcription may serve an equally vital role inregulating vascular EC and SMC proliferation. Furthermore, whilemyocardin has been shown to decrease with acute, mechanical vascularinjury and KLF4 increases, this process provides evidence that thesetranscription factors are differentially regulated in a model thatmimics early atherogenic events. Regulation in vivo in atherosclerosisis currently unknown.

Surprisingly, SMMHC was the only SMC marker that did not follow theexpected modulation trends. This may be due to RT-PCR primer recognitionof both SMMHC isoforms (SM-1 and SM-2). Analysis of each isoformseparately may elucidate a response consistent with the other SMCmarkers. Analysis at later time points (i.e., forty-eight hours) mayresolve this. The combined phenotypic responses of both ECs and SMCs inthe presence of atheroprone flow are strikingly similar to historical ECand SMC phenotype profiles defined in human and experimental models ofatherosclerosis (FIG. 10).

Evaluation of EC gene expression in response to atheroprone relative toatheroprotective flow is consistent with the only EC monoculture studyusing similar flow profiles as well as studies using similar magnitudesof steady shear stress and in vivo models of atherosclerosis,emphasizing that hemodynamics more robustly regulate the EC phenotypethan the presence of SMCs. ECs exposed to twenty-four hours ofatheroprone flow induced higher levels of proatherogenic andproliferative genes and proteins for IL-8, VCAM-1, and PCNA commensuratewith reductions in eNOS, Tie2, and KLF2. The expression loss of eNOS andTie2 suggests higher rates of remodeling and increased permeability,characteristic features of atherosusceptible regions in vivo. Evidencehas established the role of KLF2, and possibly KLF4, as an upstreamtranscriptional regulator of atheroprotection. Atheroprotectivehemodynamics in vitro and regions in vivo appear to be a key modulatorof KLF2 expression and transcriptional control. SMCs also exhibited anearly inflammatory response to atheroprone flow, as indicated byincreased VCAM-1 mRNA levels. VCAM-1 modulation has been observed inSMCs of human atherosclerotic plaques and has been linked toproliferation during early atherogenesis in vitro and in vivo. However,since the proliferative marker PCNA showed no change in SMCs foratheroprone flow, it is possible that a more migratory SMC phenotype ispresent in this system.

The EC-secreted cytokine(s)/mitogen(s) that regulates SMC phenotypicmodulation during early atherogenesis has yet to be elucidated andincludes candidates such as PDGF-BB, IL-1, and IL-8. Here, we show ECsincrease IL-8 mRNA production and IL-8 secretion following atheroproneflow. Indeed, IL-8 can stimulate the induction of a migratory phenotypein SMCs. Therefore, IL-8 secretion by ECs may be one mechanism by whichSMCs regulate a more synthetic phenotype. Of interest, a recent study inapolipoprotein E^(−/−) mice showed that experimentally induced low shearstress resulted in an increase in growth-related protein (Gro)-α mRNA.However, given the in vivo nature of this study, it was not determinedwhether changes in Gro-α mRNA were in ECs, SMCs, or both. Although Gro-αbinds the same receptors as IL-8, no murine homolog of IL-8 exists. Thehuman coculture model is therefore ideal for examining the role ofEC-derived IL-8 on SMCs, and future studies are ongoing to establish therelative contributions of such cross-communication mechanisms.

Cell morphology changes observed in atheroprone versus atheroprotectiveflow were also signs of early remodeling that could lead to localizeddownstream atherogenic responses. ECs are known to reorient in thedirection of flow under pulsatile physiologic conditions and maintain amore polygonal shape after exposure to disturbed flow, as observed inour system. However, our understanding of SMC reorientation due to shearstress sensed by the endothelium is in its earliest stages. SMCs orientmore perpendicular to hemodynamic flow under the atheroprotectivewaveform, whereas SMCs exposed to atheroprone flow resulted in morerandom alignment. Importantly, this SMC orientation is nearly identicalto the spatial patterning of SMCs in an intact blood vessel atbifurcating regions, regions highly susceptible to atherosclerosis.Together, this suggests that hemodynamic flow can regulate both EC andSMC orientation by unique control mechanisms inherent to distinctatheroprone or atheroprotective flow patterns.

This invention presents a novel in vitro coculture model using human ECsand SMCs that shows that human hemodynamic forces, atheroprotective oratheroprone, applied directly to the endothelium can modulate the SMCphenotype and influence SMC remodeling, a process we defined asmechanotranscriptional coupling. Moreover, the snapshot of phenotypicand morphologic alterations in ECs and SMCs indicates that hemodynamicforces on the endothelium are an important modulator of atherogenesis.

As shown in FIG. 1, a TRANSWELL 100 is used in the hemodynamic flowprocess. The TRANSWELL allows multiple cells 110, 120 to be tested inparallel and also provides a porous interface. An exemplary process forplating to coculture is also shown; however, this process may be alteredby processes available to one skilled in the art. In this embodiment,SMCs 110 are plated at an initial time, after which the TRANSWELL isinverted. The SMCs 110 are incubated for twenty-four to forty-eighthours, after which ECs 120 are plated on the TRANSWELL and incubated.The bottom of the Petri dish into which the TRANSWELL is inserted mayalso serve as a third surface to plate an additional cell type or thesame cell type as ones plated directly on the TRANSWELL membrane 170.After the ECs 120 are incubated, the cone 140 of the motor and conedevice is used to apply a shear force.

As shown in FIG. 2, a motor and cone device 200 is used to apply theshear forces upon the cells. A motor 230 causes the cone 240 to rotateat a precise rotational velocity, and can effect the rotation in eitherdirection (i.e. clockwise or counterclockwise). This rotational force isapplied to a liquid medium by the cone. In turn, this medium appliesshear forces directly to the cells 260 on the TRANSWELL membrane 270 inthe culture plate 250. Software is programmed to control the continuousmotion of the cone. This software file is uploaded to a motor controllerunit, and the information is then sent directly to the motor to performthe programmed task.

In a preferred embodiment, the medium is a cell culture broth that isformulated to sustain the integrity and health of the cells during theexperiment. The formulation is not limited and may vary depending on thecell types being use and experimental study. Additionally, drugcompounds may be a part of this formulation either initially, orperfused into the cell culture environment during the course of a flowexperiment. This may include, but is not limited to compound that caninhibit, activate or alter the function of proteins/genes in the cells.

In one embodiment, the device can be used to test the compatibility,cellular adhesion, and phenotypic modulation of cells from vascularstent material under hemodynamic conditions. For example endothelialand/or smooth muscle cells may be seeded next to, on top of, orunderneath the material, mounted on the stationary surface of thedevice. Materials include but are not limited to metallic nanoporousmetals, polymers, biodegradable polymers, carbon surfaces, scratched oretched surfaces. These materials further include non-degradable polymeror co-polymer, such as polyethylene-co-vinyl acetate (PEVA) and polyn-butyl methacrylate, and can be coated onto the TRANSWELL surface.These materials further include biodegradable polymer or co-polymer,such as polylactic acid glycolic acid (PLGA) or phosphorylcholine, andcan be coated onto the TRANSWELL surface. These materials furtherinclude nanoporous surface modification, such as a ceramic, metal orother material and can be added to the TRANSWELL surface as a nonporoussurface modification. These materials further include microporoussurface modification, such as a ceramic, metal, physical etching (suchas sand blasting) or other material added to the TRANSWELL surface toform a microporous surface modification.

In another embodiment of this invention, the device can operate withcells plated on either one or both sides of the TRANSWELL membrane. Themembrane portion of the TRANSWELL membrane can comprise any biologicalor synthetic material, with a range of porosities and thicknesses.Similarly, the structure that holds and supports the TRANSWELL membranecan be made of any synthetic material.

EXAMPLE

The following is an example of a method of using the present invention,and is not intended to limit the scope of the invention to the exactmethod described in this example.

Human Cell Isolation and Culture.

Primary human ECs and SMCs were isolated from umbilical cords, expanded,and used as cell sources. Human ECs were isolated from the umbilicalvein (human umbilical vein ECs) as previously described, followed byisolation of SMCs from the vein using a similar method as previouslydescribed.

ECs were used for experimentation at passage 2 and SMCs were forexperimentation used up to passage 10, both of which have beenestablished to retain the basal EC/SMC phenotype based on the retentionof specific EC and SMC markers. Cell types were separately cultured andpassaged using medium 199 (M199; BioWhitaker) supplemented with 10% FBS(GIBCO), 2 mM L-glutamine (BioWhitaker), growth factors [10 μg/mlheparin, (Sigma), 5 μg/ml endothelial cell growth supplement (Sigma),and 100 U/ml penicillin-streptomycin (GIBCO)].

TRANSWELL Coculture Plating Conditions.

As shown in FIG. 1, porous TRANSWELL membranes (polycarbonate, 10 μmthickness and 0.4 μm pore diameter, no. 3419, Corning) were initiallycoated with 0.1% gelatin on the top and bottom surfaces. The TRANSWELLwas inverted, and SMCs were plated at a density of 10,000 cells/cm² onthe bottom surface for 2 h. The TRANSWELL was then turned back over intothe holding well for forty-eight hours in reduced serum growth medium(M199 supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mlpenicillin-streptomycin). ECs were then plated on the top surface of themembrane at a density of 80,000 cells/cm² under the same mediaconditions for an additional twenty-four hours. For hemodynamic flowexperiments, two dishes were prepared in parallel.

Coculture Hemodynamic Flow Device and Flow Patterns.

As shown in FIG. 2, the novel coculture in vitro model of this processuses arterial flow patterns modeled from the human circulation wereapplied to human ECs. A version of the cone and plate device is a directdrive, whereby the cone is directly driven by the motor (rather than offto one side through a timing belt connection). This model was modifiedto incorporate a 75-mm-diameter TRANSWELL coculture dish (polycarbonate,10 μm thickness and 0.4 μm pore diameter, Corning). Additionalmodifications included a base to securely hold the TRANSWELL dish, asmaller cone (71.4 mm diameter and 1° cone angle) to fit inside theTRANSWELL compartment, and special mounting brackets for in-flow andout-flow tubing for both the inner and outer chambers of the TRANSWELL,which provides direct access to the culture fluid environment tocontinuously exchange media to both EC and SMC layers. Through therotation of the cone, the system imposes hemodynamic shear stress on theEC layer of the EC/SMC coculture.

Hemodynamic flow patterns used in this process were derived from MRI ofthe human common carotid artery (CCA) and internal carotid sinus (ICS)to best simulate atheroprotective (CCA) and atheroprone (ICS) shearstress patterns in vitro, respectively. The two hemodynamic flowconditions were run in parallel for each EC/SMC subpopulation. FIG. 3shows human hemodynamic flow profiles (left) from the common carotid(CCA; atheroprotective, right, 310) and internal carotid sinus (ICS;atheroprone, 320) were imposed on the EC surface of the TRANSWELL.

Real-Time RT-PCR.

After the application of hemodynamic flow patterns for twenty-fourhours, SMCs and ECs were rinsed two times in PBS with Ca²⁺/Mg²⁺. Themembrane was removed from the holding dish and inverted. SMCs weregently scraped toward the center of the dish with small flexible cellscrapers. Cells were then rinsed onto a sterile surface using 1 ml PBS,which was then transferred to a microcentrifuge tube on ice. Themembrane was turned over and placed flat on a sterile surface, and ECswere scraped in 1 ml PBS and then transferred to a separatemicrocentrifuge tube on ice. Tubes were centrifuged, and PBS wasremoved. Total RNA was extracted using TRIZOL reagent (Invitrogen) (amonophasic solution of phenol and guanidine isothiocyanate) and reversetranscribed using the ISCRIPT cDNA Synthesis Kit (Bio-Rad). Primers weredesigned using BEACON DESIGNER 2.0 (primer design software) for smoothmuscle α-actin (SMαA), myocardin, smooth muscle myosin heavy chain(SMMHC), VCAM-1, monocyte chemoattractant protein-1 (MCP-1), endothelialnitric oxide synthase (eNOS), angiopoiten receptor Tie2, IL-8, andKruppel-like transcription factors (KLF2 and KLF4). Table 1 shows senseand antisense primers used for each human gene. The expression of mRNAwas analyzed via real-time RT-PCR using AMPLITAQ GOLD (a modified TaqDNA polymerase that is activated when the reaction reaches an optimalannealing temperature) (Applied Biosystems), SYBR GREEN (a specificdouble-stranded DNA binding dye used to detect PCR product as itaccumulates during PCR cycles) (Invitrogen), and an ICYCLER (a real-timePCR detection system) (Bio-Rad).

TABLE 1 RT-PCR primers designed for gene and ChIP analysesSense Primer (SEQ ID NO.) Antisense Primer (SEQ ID NO.)Real-time RT-PCR primers β₂-Microglobulin 5′-AGCATTCGGGCCGAGATGTCT-3′(1)5′-CTGCTGGATGACGTGAGTAAACCT-3′(15) eNOS 5′-CTCCATTAAGAGGAGCGGCTC-3′(2)5′-CTAAGCTGGTAGGTGCCTGTG-3′(16) IL-8 5′-CATGACTTCCAAGCTGGCCG-3′(3)5′-TTTATGAATTCTCAGCCCTC-3′(17) KLF2 5′-GCACCGCCACTCACACCTG-3′(4)5′-CCGCAGCCGTCCCAGTTG-3′(18) KLF4 5′-GGCCAGAATTGGACCCGGTGTAC-3′(5)5′-GCTGCCTTTGCTGACGCTGATGA-3′(19) MCP-1 5′-CCAGCAGCAAGTGTCCCAAAG-3′(6)5′-TGCTTGTCCAGGTGGTCCATG-3′(20) Myocardin 5′-TGCAGCTCCAAATCCTCAGC-3′(7)5′-TCAGTGGCGTTGAAGAAGAGTT-3′(21) SMαA 5′-CACTGTCAGGAATCCTGTGA-3′(8)5′-CAAAGCCGGCCTTACAGA-3′(22) SMMHC 5′-AGATGGTTCTGAGGAGGAAACG-3′(9)5′-AAAACTGTAGAAAGTTGCTTATTCACT-3′(23) Tie25′-CCGTTAATCACTATGAGGCTTGGC-3′(10) 5′-GTGAAGCGTCTCACAGGTCCA-3′(24)VCAM-1 5′-GTTTGTCAGGCTAAGTTACATATTGATGA-3′(11)5′-GGGCAACATTGACATAAAGTGTTT-3′(25) ChIP analysis primers SMαA, 5′-CArG5′-AGCAGAACAGAGGAATGCAGTGGAAGAGAC-3′(12)5′-CCTCCCACTCGCCTCCCAAACAAGGAGC-3′(26) SMMHC, 5′-CArG5′-CTGCGCGGGACCATATTTAGTCAGGGGGAG-3′(13)5′-CTGGGCGGGAGACAACCCAAAAAGGCCAGG-3′(27) c-fos5′-CCCGCACTGCACCCTCGGTG-3′(14) 5′-TACAGGGAAAGGCCGTGGAAACCTG-3′(28) ChIP,chromatin immunoprecipitation; eNOS, endothelial nitric oxide synthase;KLF, Kruppel-like factor. MCP, monocyte chemoattractant protein: SMαA,smooth muscle α-actin: SMMHC, smooth muscle myosin heavy chain; CArG,CC(A/T)₆GG.

Western Blot Analysis.

Vascular SMCs and ECs were collected as described in Real-time PCR andlysed in RIPA buffer (1% Nonidet P-40, Na-deoxycholate, 1 mM EDTA, 1 mMPMSF, 1 mM Na3VO4, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1μg/ml pepstatin). Total protein lysates were resolved on a 7.5% SDS-PAGEgel and blotted on a polyvinyl derivative membrane. Primary antibodies[SMαA (Sigma, 1:1,000), eNOS (BD Transduction Laboratories, 0.1 μg/ml),VCAM-1 (R&D Systems, 1:500), and PCNA (Cell Signaling, 1:1,000)] wereincubated with the blot for one hour at room temperature or overnight at4° C. Horseradish peroxidase-conjugated secondary antibodies [goatanti-rabbit, goat anti-mouse (Santa Cruz Biotechnology, 1:5,000), anddonkey anti-goat (1:5,000)] were incubated with the blot for one hour atroom temperature. An ALPHAIMAGER 8900 (a gel imaging system) andALPHAEASEFC software (image analysis software) were used for acquisitionof blot image and densitometry analysis, respectively.

ELISA.

Cocultured TRANSWELLS were prepared and exposed to differentialhemodynamic environments. Media perfused throughout the flow experimentwere collected on ice after 4, 8, 12, and 24 h for each chamber of themembrane (i.e., EC— and SMC-conditioned media from atheroprone andatheroprotective flows). Samples were then stored at −80° C. until theywere assayed for IL-8 secreted protein via ELISA (GE Healthcare). Theconcentration of protein was determined using a spectrophotometer at 450nm and normalized to the volume of media collected per hour.

Chromatin Immunoprecipitation Assay.

After the application of flow patterns, chromatin immunoprecipitation(ChIP) was performed as previously described with modifications allowingfor a quantitative analysis of protein:DNA interactions. Outflow mediafrom each experiment were supplemented with 1% formaldehyde and thenincubated with cells for 10 min immediately following 24 h of flow.Antibodies included rabbit polyclonal anti-serum response factor (SRF;Santa Cruz Biotechnology, 5 μg/ml) and anti-histone H4 acetylation(Upstate Biotechnologies, 5 μg/ml). Recovered DNA was quantified byfluorescence with PICOGREEN reagent (a fluorescent nucleic acid stainfor quantifying double-stranded DNA) (Molecular Probes) according to themanufacturer's recommendations. Real-time PCR was performed on 1 nggenomic DNA from ChIP experiments with minor modifications as previouslydescribed. Real-time PCR primers were designed to flank the5′-CC(a/T)₆GG (CArG) elements of SMαA, SMMHC, c-fos CArG. Table 1 showsthe primers used for ChIP analysis. Quantification of protein:DNAinteraction/enrichment was determined by the following equation: 2^((C)^(t Ref) ^(−C) ^(t IP)) −2^((C) ^(t Ref) ^(−C) ^(t No antibody control)), where C_(t Ref) is the reference threshold cycle (C_(t)) and C_(t ip)is the C_(t) of the immunoprecipitate. ChIP data are representative offive to six independent experiments pooled together and analyzed induplicate.

Immunofluorescence.

For immunofluorescence (IF), TRANSWELL membranes were fixed in 4%paraformaldehyde for both en face preparations and transverse sections.En face preparations were permeabilized in 0.2% Triton X-100. Primaryantibody for SMCs was pipetted onto a piece of PARAFILM (self-sealing,moldable and flexible film) [Cy3-SMαA (Sigma, 4 μg/ml) and SMMHC(Biomedical Technologies, 1:100)], and the sample well was placed ontop. Primary antibody for ECs [vascular endothelial cadherin (VE-cad;Santa Cruz Biotechnology, 2 μg/ml)] was then added directly to theinside of the well, and both antibodies were simultaneously incubatedfor one hour. Similarly, secondary antibodies [Cy2 donkey anti-goat(Jackson ImmunoResearch, 4 μg/ml) and ALEXA FLUOR 546 (a fluorescent dyewith an orange emission color) goat anti-rabbit (Molecular Probes, 6μg/ml)] were added to samples as required and incubated for 1 h. Sampleswere mounted by adding PROLONG GOLD Antifade Reagent (an antifadereagent) with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes) toa large coverslip and dropping the well on top. Another drop of DAPI wasadded to the inside of the well, and a 22-mm-diameter coverslip wasplaced on top and allowed to solidify. The holding well was removed fromthe mounted samples using a scalpel to allow for imaging. Confocalmicroscopy was used to image en face samples through the z-axis from theEC to SMC layer (Nikon ECLIPSE Microscope TE2000-E2 and Melles GriotArgon Ion Laser System no. 35-IMA-840).

To prepare the transverse sections, EC/SMC cultures were stained withphalloidin-488 (Molecular Probes) or FM 4-64FX (Molecular Probes) usingthe methodology described above, immersed in 30% sucrose overnight,frozen in OCT compound, sliced into 5-μm-thick sections with a cryostat,and then mounted for assessment by confocal microscopy. IF stainedsamples were analyzed using a confocal microscope and differentialinterference contrast for cell-to-cell interactions within the pores ofthe TRANSWELL membrane under static conditions, as previously described.

EC/SMC Orientation and Morphometric Measurements.

The orientation of ECs and SMCs relative to the direction of flow wasquantified using confocal microscopy of IF stained samples. Followinghemodynamic flow, the coculture was fixed as described above, andisosceles triangular samples from the 75-mm-diameter dishes were cutwith the apex of the triangle pointing toward the center of the dish.This method established the correct orientation relative to thedirection of flow. Samples were then stained as described above andmounted between two coverslips. For imaging, samples were oriented onthe confocal stage with the triangle apex facing to the right, so thatthe direction of flow was consistent across all samples. Images weretaken of ECs and SMCs in the same location, separated only by themembrane distance.

At least three microscopy fields were acquired over three independentexperiments. METAMORPH software (i.e., image acquisition and analysissoftware) was used to determine the angle of orientation and shapefactor (SF) for each cell analyzed relative to the direction of flow. Todetermine the elongation of cell types, borders stained for VE-cad (FIG.6) and β-catenin (not shown) of ECs (CCA: n=111 and ICS: n=53) and SMαA(FIG. 6), SMMHC, and β-catenin (not shown) of SMCs (CCA: n=64 and ICS:n=25) were outlined, and measurements of the area and perimeter wereoutputted. SF was calculated using the following equation: SF=(4πA)/P²,where A is the cell area and P is the perimeter. For each SF bin in thehistogram range, the number of cells per bin was normalized to the totalnumber of cells analyzed over the whole range to yield a normalizedfrequency. Histograms were plotted to show the distribution of SFs foreach condition (see FIG. 7). For the angle of orientation, lines weredrawn in both the direction of flow and along the long axis of the SMCsfrom both flow patterns (CCA: n=119 and ICS: n=104) and ECs foratheroprotective flow only (CCA: n=124). The angle between the two lineswas measured as the orientation angle relative to the flow direction,and histograms were plotted so that the frequency of cells having thesame orientation was represented as the bar length.

Data Analysis and Statistics.

Real-time RT-PCR results are reported as the fold induction of cycleamplification times for atheroprone flow samples compared withatheroprotective flow and normalized to endogenously expressed geneβ₂-microglobulin. Student's t-test was conducted for mRNA, orientation,and elongation data to determine the significance in expression level ormorphological changes as a function of hemodynamic flow pattern andtime. Data from at least three independent experiments per conditionwere used for analysis and evaluated at P<0.05.

Exemplary Results

Optimization of EC/SMC Coculture Plating and Growth Conditions.

Coculture conditions for human EC and SMC plating were optimized so thateach cell type reached confluence prior to the application ofhemodynamic flow. FIG. 4 shows confluent layers of ECs and SMCstwenty-four hours following EC seeding. More specifically, FIG. 4 showsECs (left) and SMCs (right) cocultured for twenty-four hours showingconfluency status (Top, en face images; bottom, transverse section). ECsretained their classic polygonal morphology, forming adherens junctions,as demonstrated by the continuous peripheral staining of VE-cad, whereasSMCs were elongated and randomly oriented in the typical “hill andvalley” formation. In SMCs plated alone, reduced serum media (2% FBScompared with 10% FBS) increased the mRNA expression of SMC markers SMαAand myocardin, indicating a more differentiated SMC phenotype(normalized gene expression with 2% FBS: SMA, 2.51±0.36 and myocardin,2.07±0.05; with 10% FBS: SMA, 0.69±0.23 and myocardin, 0.54±0.14; seeFIG. 10).

A murine coculture model has recently demonstrated that ECs and SMCsphysically interact and communicate via gap junctions through linearpores of the TRANSWELL membrane. This model emulates myoendothelialjunctions present within the vascular wall in vivo, creating a means forionic communication via gap junctions and physical heterocellularadhesion. To determine whether EC/SMC physical interactions are formedin our human coculture model, transverse sections of the TRANSWELLmembrane were IF labeled for F-actin or FM 4-64FX and analyzed usingconfocal and phase contrast microscopy. The results shown in FIG. 5demonstrate that cellular processes are present in the pores,establishing heterocellular interactions. Transverse sections arestained for F-actin (top) and FM 4-64 (middle) or visualized bydifferential interference contrast (bottom) and showed cellularprocesses within membrane pores 510, 520, 530. Shown are representativeimages from three independent experiments. Bars on en face images equal50 μm; bars on transverse sections equal 10 μm.

EC/SMC Morphological Remodeling is Altered in Atheroprone Flow.

The morphology of ECs and SMCs in vivo is highly ordered, with ECs beingelongated and aligned with the direction of hemodynamic flow and SMCsoriented perpendicular to the long axis of the artery and direction ofblood flow. However, the endothelium in regions of complex flow, such asin arterial bifurcations, is more polygonal and less aligned, and SMCsdo not consistently align perpendicular to flow. To determine whetherhemodynamic flow on the endothelium induces morphological changes to ECsand SMCs, the following SF measurements for both cell types weredetermined: 1) alterations in elongation and 2) orientation anglemeasurements relative to the direction of flow. Significant differencesin both cell shape (SF) and cell orientation were observed after theapplication of atheroprone flow compared with atheroprotective flow asshown in FIGS. 6-8. SF indicates the extent of cellular elongation,where a value of 1 specifies a circle (i.e., no elongation) and a valuecloser to 0 specifies an elongated cell. Representative IF images areshown in FIG. 6. As previously established, ECs exposed to atheroproneflow maintained a more polygonal shape (SF=0.75±0.002), whereas ECsunder atheroprotective conditions were more elongated (SF=0.64±0.015).EC/SMC morphology and orientation were determined by immunofluorescencefollowing flow. ECs were stained for vascular endothelial cadherin(VE-cadherin) and SMCs were stained for smooth muscle α-actin (SMαA).The arrow in FIG. 6 indicates the direction of net flow and the barsequal 50 p.m.

FIG. 7 shows the distribution of EC SF normalized to the number of cellsanalyzed. The alignment of ECs coincided with the direction of flow whenexposed to atheroprotective flow (angle relative to flow=8.6±4.01°; FIG.8), whereas no preferential polarity of ECs under atheroprone flow couldbe measured due to the rounded morphology.

SMCs on the TRANSWELL exposed to atheroprone flow showed a significantbut small increase in elongation (SF=0.26±0.009) than those exposed toatheroprotective flow (SF=0.31±0.018; FIGS. 6 and 7). Interestingly,SMCs in atheroprotective flow consistently aligned more toward aperpendicular orientation relative to the direction of flow (FIGS. 8 and9), whereas, in contrast, SMCs under atheroprone conditions exhibited amore random, less coordinated orientation (−47.9±1.3° vs. −13.1±5.0°,respectively, P<0.0001). FIG. 6 shows representative images of SMCorientation relative to flow, and FIG. 9 shows the histogramdistribution of SMC orientation.

Purity of RNA and Protein Isolation from ECs/SMCs Following HemodynamicFlow.

The purity of collected RNA and protein from each cell layer followingthe flow experiment was assessed by real-time RT-PCR and Western blotanalysis for the presence of EC- and SMC-specific proteins (eNOS andSMαA, respectively; FIG. 11 and FIG. 12). No cross-contamination at themRNA or protein level was detectable.

FIG. 11 shows real-time RT-PCR on EC and SMC populations followingtwenty-four hours of atheroprotective flow. Both cell types expressedrespective SMC and EC markers [SMαA and endothelial nitric oxide synthse(eNOS), respectively] after the isolation of each cell type. SMCsexpressed significantly larger quantities of SMαA than ECs, and the ECexpression of eNOS was significantly greater than that of SMCs after CCAflow, showing that the populations of cells analyzed for differentialgene regulation were pure. Values are mean±SE; n=3; *P>0.05.

FIG. 12 shows protein analysis confirming that only SMCs express SMαAand only ECs express eNOS. IB, immunoblot analysis.

Atheroprone Flow Differentially Regulates EC and SMC Phenotypes andPromotes Proinflammatory Priming.

The major goal was to determine whether differential humanderivedhemodynamic flow patterns applied to ECs influence SMC phenotypicmodulation. Given this objective, changes in established markersindicating EC and SMC phenotypic modulation were examined twenty-fourhours after the application of atheroprone or atheroprotective flow.Genes of interest were classified as EC- or SMC-specific cell markers(EC: eNOS, Tie2, and KLF2/KLF4; SMC: SMαA, SMMHC, and myocardin) orinflammatory markers (VCAM-1, IL-8, and MCP-1). Additionally, proteinanalysis was performed on a subset of markers (eNOS, SMαA, VCAM-1, andPCNA). Modulation of genes and proteins was determined by the relativechange in atheroprone compared with atheroprotective flow.

Consistently, significant reductions in mRNA levels of EC quiescentmarkers eNOS, Tie2, KLF2, and KLF4 were observed in response toatheroprone flow (FIG. 13), which was also confirmed by changes inprotein levels of eNOS (FIG. 15). Modulation of these EC markers haspreviously been demonstrated via shear stress stimuli relating toatherosclerosis; however, such a comprehensive examination of ECphenotype has never occurred in the presence of SMCs for hemodyanamicflow patterns.

Classic SMC differentiation markers have never before been analyzed forgene modulation in a coculture model exposed to any shear stressstimulus. Hallmarks of SMC phenotypic modulation associated withatherosclerosis included a decrease in genes defining the quiescentcontractile phenotype (e.g., SMαA, SMMHC, and myocardin), an increase ingenes associated with the synthetic phenotype (e.g., KLF4 and VCAM-1),and the initiation of proliferative and migratory events. In thepresence of atheroprone flow, SMCs showed a significant reduction in SMCdifferentiation markers SMαA and myocardin (FIG. 13). Protein analysisfurther confirmed this observation for SMαA (FIG. 15). Although thetranscription factor KLF4, which was recently discovered to be importantin suppressing myocardin-dependent transcription, was not significantlyinduced (P=0.10) for atheroprone relative to atheroprotective flow, thistrend may still point toward a mechanism of regulating SMC phenotypicswitching. Since vascular injury maximally induced KLF4 after just 4 h,it is possible that at twenty-four hours of flow, the maximal responseof KLF4 was missed. Notably, SMMHC was not significantly modulated(P=0.62).

Most interesting was that the reduction in EC quiescent markers and SMCcontractile markers corresponded with the upregulation of severalproinflammatory genes. VCAM-1 was significantly upregulated in both ECsand SMCs at both the mRNA and protein level (FIGS. 14 and 15). Asignificant increase in IL-8, a proinflammatory gene downstream of NF-κBactivation, was also observed in ECs at the mRNA level. Secretion ofIL-8 from EC and SMC layers was further measured as a function of timeduring the application of both flow patterns and was only significantlyaugmented in ECs during later time points of atheroprone flow (FIG. 16).In contrast, decreases in IL-8 and MCP-1 were concurrently observed inSMCs (FIG. 14). Finally, analysis of the proliferative marker PCNAshowed increased protein levels in ECs exposed to atheroprone flow butno change for SMCs (FIG. 15).

To control for a flow-induced EC influence on the SMC response, SMCswere plated under two conditions in monoculture: 1) on the bottom of theTRANSWELL holding dish in the presence of a TRANSWELL membrane (SMC D)or 2) on the bottom of the TRANSWELL membrane (SMC T), as shown in FIG.17. For each condition, flow was applied to the top of the TRANSWELLmembrane without ECs. Real-time RT-PCR analysis of samples showed thatsignificant differences existed between each condition for SMαA andVCAM-1 but not for myocardin (FIG. 17). VCAM-1 was the only geneappreciably inducted by atheroprone flow for both conditions. Potentialconfounding factors introduced for the SMC T condition were smoothmuscle cellular processes that extruded through the porous membrane tothe top of the TRANSWELL where flow was being applied (FIG. 18), whichwas not observed in the experiments with ECs present. The significantchanges between each condition (SMC D vs. SMC T) indicate thesensitivity of SMCs to their local environment. Thus, for this study,comparison between the two distinct flow patterns applied in thepresence of both cell types was the most robust method to control forall features (e.g., media exchange, experimental setup, time in culture,and heterocellular presence) of the hemodynamic coculture environment.

Arterial hemodynamics Control Epigentic Regulation of SMC GeneExpression.

Many of the promoter regions of genes that encode SMC-selectivecontractile proteins contain CArG cis-regulatory elements that bind SRF,including SMαA and SMMHC. ChIP experiments were conducted to determinewhether SRF binding and histone H4 acetylation in 5′-CArG promoterregions of the SMαA, SMMHC, and c-fos promoters were regulated at theepigentic level by hemodynamic flow. The results indicated a reductionof histone H4 acetylation and SRF binding in response to atheroproneflow relative to atheroprotective flow for SMαA and SMMHC (FIG. 19).Conversely, histone H4 acetylation and SRF binding to the c-fos CArGregion was not statistically different among flow conditions (FIG. 19).This epigenetic fingerprint was identical to in vitro experiments inSMCs in response to PDGF-BB and in vivo in response to acute vascularinjury.

Drugs

The drug may be selected from a group comprising actinomycin-D,batimistat, c-myc antisense, dexamethasone, paclitaxel, taxanes,sirolimus, tacrolimus and everolimus, unfractionated heparin,low-molecular weight heparin, enoxaprin, bivalirudin, tyrosine kinaseinhibitors, GLEEVEC (imatinib), wortmannin, PDGF inhibitors, AG1295, rhokinase inhibitors, Y27632, calcium channel blockers, amlodipine,nifedipine, and ACE inhibitors, synthetic polysaccharides, ticlopinin,dipyridamole, clopidogrel, fondaparinux, streptokinase, urokinase,r-urokinase, r-prourokinase, rt-PA, APSAC, TNK-rt-PA, reteplase,alteplase, monteplase, lanoplase, pamiteplase, staphylokinase,abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban, ananti-restenosis agent, an anti-thrombogenic agent, an antibiotic, ananti-platelet agent, an anti-clotting agent, an anti-inflammatory agent,an anti-neoplastic agent, an anti-hypertensive agent, a chelating agent,penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA(succimer), deferoxamine mesylate, a cholesterol lowering agent, astatin, an agent that raises HDL, a cyclyoxygenase inhibitor, CELEBREX(celecoxib), VIOXX (rofecoxib), a radiocontrast agent, a radio-isotope,a prodrug, antibody fragments, antibodies, live cells, therapeutic drugdelivery microspheres or microbeads, and any combinations thereof.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventionconcept as defined by the appended claims and their equivalents.

What is claimed is:
 1. A method of mimicking hemodynamic flow duringcell culture, said method comprising the steps of: adding a culturemedia to a Petri dish; plating a first cell type on a first side of aporous membrane; plating a second cell type on a second side of theporous membrane, wherein said porous membrane is suspended in the Petridish such that the first side is proximal and in spaced relation to asurface of the Petri dish, thereby defining within the Petri dish alower volume comprising the first cell type and an upper volumecomprising the second cell type, the porous membrane being adapted topermit fluid communication of the culture media and physical interactionand communication between cells of the first cell type and cells of thesecond cell type, and all of the cell types are within the culturemedia; perfusing culture media into and out of the upper volume throughinlets and outlets within the portion of the Petri dish defining theupper volume; perfusing culture media into and out of the lower volumethrough inlets and outlets within the portion of the Petri dish definingthe lower volume; and applying a shear force upon the plated second celltype, said shear force resulting from flow of the culture media inducedby a hemodynamic flow device, said flow mimicking hemodynamic flow;wherein at least one of the first cell type and the second cell type isa vascular cell type.
 2. The method of claim 1, further comprising thestep of culturing all of the cell types.
 3. The method of claim 1,wherein said hemodynamic flow is time-variant.
 4. The method of claim 1,wherein the hemodynamic flow is derived from a previously measuredhemodynamic pattern.
 5. The method of claim 4, wherein the previouslymeasured hemodynamic pattern is human derived.
 6. The method of claim 5,wherein said pattern is derived from a patient having a pathologicalcondition.
 7. The method of claim 4, wherein the hemodynamic pattern isfrom an artery, a vein or an organ.
 8. The method of claim 4, whereinsaid hemodynamic pattern is derived from analysis of ultrasound data. 9.The method of claim 4, wherein said hemodynamic pattern is derived fromanalysis of magnetic resonance imaging (MRI) data.
 10. The method ofclaim 1, further comprising the step of analyzing at least one of thefirst cell type and the second cell type after applying the shear forcefor a period of time.
 11. The method of claim 1, further comprisinganalyzing said culture media for cytokine or humoral factor secretion.12. The method of claim 1, wherein said first cell type is renal cells,cells of the airways, or cells of the blood-brain barrier, and saidsecond cell type is vascular cells.
 13. The method of claim 1, whereinthe first cell type is smooth muscle cells, glial cells, neurons, orepithelial podocytes.
 14. The method of claim 13, wherein the secondcell type is endothelial cells.
 15. The method of claim 13, wherein theglial cells comprise astrocytes.
 16. The method of claim 1, wherein thesecond cell type is endothelial cells.
 17. The method of claim 1,wherein at least one of the first cell type and the second cell type arevascular or organ cells from one or more patients with an identifiedgenotype linked to drug toxicity or a pathophysiological endpoint. 18.The method of claim 17, wherein said one or more patients have a singlenucleotide polymorphism linked to drug toxicity or a pathophysiologicalendpoint.
 19. The method of claim 1, further comprising either plating athird cell type on the surface of the Petri dish, or suspending a thirdcell type in the culture media in the lower volume.
 20. The method ofclaim 19, further comprising the step of culturing all of the celltypes.
 21. The method of claim 19, further comprising the step ofanalyzing at least one of the first cell type, the second cell type orthe third cell type after applying the shear force for a period of time.22. The method of claim 19, wherein the hemodynamic flow is derived froma previously measured hemodynamic pattern.
 23. The method of claim 22,wherein said hemodynamic flow is time-variant.
 24. The method of claim22, wherein the previously measured hemodynamic pattern is humanderived.
 25. The method of claim 19, wherein said hemodynamic flow istime-variant.
 26. The method of claim 19, wherein the first cell type issmooth muscle cells, glial cells, neurons, or epithelial podocytes. 27.The method of claim 26, wherein the glial cells comprise astrocytes. 28.The method of claim 19, wherein the second cell type is endothelialcells.
 29. The method of claim 19, wherein the third cell type is smoothmuscle cells, glial cells, neurons, macrophages, or leukocytes.
 30. Themethod of claim 29, wherein the glial cells comprise astrocytes.
 31. Themethod of claim 19, wherein said first cell type and said third celltype are renal cells, cells of the airways, or cells of theblood-brain-barrier, and wherein said second cell type is vascularcells.
 32. The method of claim 1, wherein the shear force is applied bya device for mimicking hemodynamic flow during cell culture, said devicecomprising: an electronic controller for receiving a set of electronicinstructions; a motor operated by the electronic controller; and a shearforce applicator operatively connected to the motor for being driven bythe motor.
 33. The method of claim 32, wherein the shear forceapplicator comprises a cone attached to the motor.
 34. The method ofclaim 32, wherein the device further comprises inlets and outlets withinthe portions of the Petri dish defining the upper and lower volumes.